Tech Bulletin

Introduction Tissue dissociation methods can cause significant amounts of cell death and alter the transcriptome. Furthermore, attaining a sample that is of sufficient quality for a sequencing experiment can be almost impossible when working with tissue samples. Using nuclei instead of intact cells has become a growing method for downstream next-generation sequencing applications, single nucleus RNA- Seq (snRNA-seq). Using nuclei expands the capability to work with preserved samples and cell types that are challenging to obtain a single cell suspension from such as neurons. However, nuclei isolation methods often leave a significant amount of debris in the suspension that can...

Plasma membrane (PM) proteins play a critical role in a variety of physiological and pathological processes. Signal transduction, molecular transport and cell-cell interactions are all mediated by PM proteins. PM proteins include a variety of important proteins such as neurotransmitter receptors, G-proteins, carriers, voltage-gated ion channels, CD antigens and many drug targets. The detection, characterization, and intracellular trafficking of PM proteins are, therefore, essential for understanding of biological systems. Isolation/purification is usually the first step for characterization and profiling of PM proteins. However, it has been proven to be particularly challenging because of their low abundance and the nature of...

The Golgi apparatus is a complex organelle found in eukaryotic cells, responsible for sorting, modifying, and transporting proteins. To understand its structure and function, Golgi isolation is necessary. Density gradient centrifugation is a commonly used method that separates the Golgi based on its density. However, this approach presents challenges, including contamination from other organelles and debris, which can interfere with further analysis. A major difficulty with density gradient centrifugation is the heterogeneity of the Golgi sub-compartments, such as the cis-Golgi, medial-Golgi, and trans-Golgi. These sub-compartments differ in density and composition, leading to overlapping gradients and complications in accurately separating and...

The Golgi apparatus is consisted of a series offlattened membrane vesicles.The major function of Golgi is to modify proteins for transportation.Modified proteins are transported out of Golgi in coated vesicles that bud off the trans‐cellular membrane. It plays a critical role in protein synthesis  and modification. There are limited selections of commercial kits for isolation of this important organelle.  Brand/manufacturer Brand# 1 Invent Biotech Product Name Golgi Isolation Kit nuteTM Golgi Apparatus Enrichment Kit Mechanism of Isolation Density gradient centrifugation Spin column-based -precipitation Average sample size Tissue (5 g) Tissue (35 mg) Cells (30 million) Number of buffers and reagents used 3 4 Buffer preparation prior to use Required Not required Homogenizer Required Not required Density gradient formation Not Required...

Lysosomes are organelles enclosed by membranes that play a role in breaking down and recycling cellular materials. However, isolating lysosomes is challenging due to their fragility, small size, and scarcity, as well as the risk of contamination from other organelles. Various methods have been developed to isolate lysosomes, each with its limitations. Density gradient centrifugation is a widely used method where lysosomes are separated based on density. Unfortunately, this approach can lead to contamination with similar organelles in buoyant density, such as mitochondria or peroxisomes. Moreover, lysosomes are highly sensitive to pH and osmolarity changes, which can cause them to...